Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay.

Nontyphoidal salmonellosis is one of the most significant foodborne diseases in the United States and globally. There are no vaccines available for human use to prevent this disease, and only broad-spectrum antibiotics are available to treat complicated cases of the disease. However, antibiotic resistance is on the rise and new therapeutics are needed. We previously identified the Salmonella fraB gene, that mutation of causes attenuation of fitness in the murine gastrointestinal tract. The FraB gene product is encoded in an operon responsible for the uptake and utilization of fructose-asparagine (F-Asn), an Amadori product found in several human foods. Mutations in fraB cause an accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp), which is toxic to Salmonella. The F-Asn catabolic pathway is found only in the nontyphoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium; it is not found in humans. Thus, targeting FraB with novel antimicrobials is expected to be Salmonella specific, leaving the normal microbiota largely intact and having no effect on the host. We performed high-throughput screening (HTS) to identify small-molecule inhibitors of FraB using growth-based assays comparing a wild-type Salmonella and a Δfra island mutant control. We screened 224,009 compounds in duplicate. After hit triage and validation, we found three compounds that inhibit Salmonella in an fra-dependent manner, with 50% inhibitory concentration (IC50) values ranging from 89 to 150 µM. Testing these compounds with recombinant FraB and synthetic 6-P-F-Asp confirmed that they are uncompetitive inhibitors of FraB with Ki' (inhibitor constant) values ranging from 26 to 116 µM. IMPORTANCE Nontyphoidal salmonellosis is a serious threat in the United States and globally. We have recently identified an enzyme, FraB, that when mutated renders Salmonella growth defective in vitro and unfit in mouse models of gastroenteritis. FraB is quite rare in bacteria and is not found in humans or other animals. Here, we have identified small-molecule inhibitors of FraB that inhibit the growth of Salmonella. These could provide the foundation for a therapeutic to reduce the duration and severity of Salmonella infections.

Serendipitous Discovery of a Competitive Inhibitor of FraB, a Salmonella Deglycase and Drug Target.

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 µM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

Characterization of a Salmonella Transcription Factor-DNA Complex and Identification of the Inducer by Native Mass Spectrometry.

FraR, a transcriptional repressor, was postulated to regulate the metabolism of the Amadori compound fructose-asparagine (F-Asn) in the foodborne pathogen Salmonella enterica. Here, the DNA- and inducer-binding affinities and stoichiometries of FraR were determined and cross-validated by electrophoretic mobility-shift assays (EMSAs) and online buffer exchange coupled to native mass spectrometry (OBE-nMS). We demonstrate the utility of OBE-nMS to characterize protein and protein-DNA complexes that are not amenable to offline exchange into volatile buffers. OBE-nMS complemented EMSAs by revealing that FraR binds to the operator DNA as a dimer and by establishing 6-phosphofructose-aspartate as the inducer that weakens DNA binding by FraR. These results provide insights into how FraR regulates the expression of F-Asn-catabolizing enzymes and add to our understanding of the intricate bacterial circuitry that dictates utilization of diverse nutrients.

Synthesis and crystal structures of 3,6-di-hydroxy-picolinic acid and its labile inter-mediate dipotassium 3-hy-droxy-6-(sulfonato-oxy)pyridine-2-carboxyl-ate monohydrate.

A simplified two-step synthesis of 3,6-di-hydroxy-picolinic acid (3-hy-droxy-6-oxo-1,6-di-hydro-pyridine-2-carb-oxy-lic acid), C6H5NO4 (II), an inter-mediate in the metabolism of picolinic acid, is described. The crystal structure of II, along with that of a labile inter-mediate, dipotassium 3-hy-droxy-6-(sulfonato-oxy)pyridine-2-carboxyl-ate monohydrate, 2K+·C6H3NO7S2-·H2O (I), is also described. Compound I comprises a pyridine ring with carboxyl-ate, hydroxyl (connected by an intra-molecular O-H⋯O hydrogen bond), and sulfate groups at the 2-, 3-, and 6-positions, respectively, along with two potassium cations for charge balance and one water mol-ecule of crystallization. These components are connected into a three-dimensional network by O-H⋯O hydrogen bonds arising from the water mol-ecule, C-H⋯O inter-actions and π-π stacking of pyridine rings. In II, the ring nitro-gen atom is protonated, with charge balance provided by the carboxyl-ate group (i.e., a zwitterion). The intra-molecular O-H⋯O hydrogen bond observed in I is preserved in II. Crystals of II have unusual space-group symmetry of type Abm2 in which extended planar networks of O-H⋯O and N-H⋯O hydrogen-bonded mol-ecules form sheets lying parallel to the ac plane, constrained to b = 0.25 (and 0.75). The structure was refined as a 50:50 inversion twin. A minor disorder component was modeled by reflection of the major component across a mirror plane perpendicular to c.

4,15-Dimethyl-7,12-diazo-niatri-cyclo-[10.4.0.02,7]hexa-deca-1(12),2,4,6,13,15-hexa-ene dibromide monohydrate.

The title compound, C16H20N2 2+·2Br-·H2O (1) is a member of the class of compounds called viologens. Viologens are quaternary salts of di-pyridyls and are especially useful as redox indicators as a result of their large negative one-electron reduction potentials. Compound 1 consists of a dication composed of a pair of 4-methyl-pyridine rings mutually joined at the 2-position, with a dihedral angle between the pyridine rings of 62.35 (4)°. In addition, the rings are tethered via the pyridine nitro-gen atoms by a tetra-methyl-ene bridge. Charge balance is provided by a pair of bromide anions, which are hydrogen bonded to a single water mol-ecule [D O⋯Br = 3.3670 (15) and 3.3856 (15) Å]. The crystal structure of 1, details of an improved synthesis, and a full analysis of its NMR spectra are presented.

MtcB, a member of the MttB superfamily from the human gut acetogen Eubacterium limosum, is a cobalamin-dependent carnitine demethylase.

The trimethylamine methyltransferase MttB is the first described member of a superfamily comprising thousands of microbial proteins. Most members of the MttB superfamily are encoded by genes that lack the codon for pyrrolysine characteristic of trimethylamine methyltransferases, raising questions about the activities of these proteins. The superfamily member MtcB is found in the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that can grow by demethylation of l-carnitine. Here, we demonstrate that MtcB catalyzes l-carnitine demethylation. When growing on l-carnitine, E. limosum excreted the unusual biological product norcarnitine as well as acetate, butyrate, and caproate. Cellular extracts of E. limosum grown on l-carnitine, but not lactate, methylated cob-(I)alamin or tetrahydrofolate using l-carnitine as methyl donor. MtcB, along with the corrinoid protein MtqC and the methylcorrinoid:tetrahydrofolate methyltransferase MtqA, were much more abundant in E. limosum cells grown on l-carnitine than on lactate. Recombinant MtcB methylates either cob(I)alamin or Co(I)-MtqC in the presence of l-carnitine and, to a much lesser extent, γ-butyrobetaine. Other quaternary amines were not substrates. Recombinant MtcB, MtqC, and MtqA methylated tetrahydrofolate via l-carnitine, forming a key intermediate in the acetogenic Wood-Ljungdahl pathway. To our knowledge, MtcB methylation of cobalamin or Co(I)-MtqC represents the first described mechanism of biological l-carnitine demethylation. The conversion of l-carnitine and its derivative γ-butyrobetaine to trimethylamine by the gut microbiome has been linked to cardiovascular disease. The activities of MtcB and related proteins in E. limosum might demethylate proatherogenic quaternary amines and contribute to the perceived health benefits of this human gut symbiont.

MtpB, a member of the MttB superfamily from the human intestinal acetogen Eubacterium limosum, catalyzes proline betaine demethylation.

The trimethylamine methyltransferase MttB is the founding member of a widely distributed superfamily of microbial proteins. Genes encoding most members of the MttB superfamily lack the codon for pyrrolysine that distinguishes previously characterized trimethylamine methyltransferases, leaving the function(s) of most of the enzymes in this superfamily unknown. Here, investigating the MttB family member MtpB from the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that excretes N-methyl proline during growth on proline betaine, we demonstrate that MtpB catalyzes anoxic demethylation of proline betaine. MtpB along with MtqC (a corrinoid protein) and MtqA (a methylcorrinoid:tetrahydrofolate methyltransferase) was much more abundant in E. limosum cells grown on proline betaine than on lactate. We observed that recombinant MtpB methylates Co(I)-MtqC in the presence of proline betaine and that other quaternary amines are much less preferred substrates. MtpB, MtqC, and MtqA catalyze tetrahydrofolate methylation with proline betaine, thereby forming a key intermediate in the Wood-Ljungdahl acetogenesis pathway. To our knowledge, MtpB methylation of Co(I)-MtqC for the subsequent methylation of tetrahydrofolate represents the first described anoxic mechanism of proline betaine demethylation. The activities of MtpB and associated proteins in acetogens or other anaerobes provide a possible mechanism for the production of N-methyl proline by the gut microbiome. MtpB's activity characterized here strengthens the hypothesis that much of the MttB superfamily comprises quaternary amine-dependent methyltransferases.

Relative interfacial cleavage energetics of protein complexes revealed by surface collisions.

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

Measurement of Fructose-Asparagine Concentrations in Human and Animal Foods.

The food-borne bacterial pathogen, Salmonella enterica, can utilize fructose-asparagine (F-Asn) as its sole carbon and nitrogen source. F-Asn is the product of an Amadori rearrangement following the nonenzymatic condensation of glucose and asparagine. Heating converts F-Asn via complex Maillard reactions to a variety of molecules that contribute to the color, taste, and aroma of heated foods. Among these end derivatives is acrylamide, which is present in some foods, especially in fried potatoes. The F-Asn utilization pathway in Salmonella, specifically FraB, is a potential drug target because inhibition of this enzyme would lead to intoxication of Salmonella in the presence of F-Asn. However, F-Asn would need to be packaged with the FraB inhibitor or available in human foods. To determine if there are foods that have sufficient F-Asn, we measured F-Asn concentrations in a variety of human and animal foods. The 400 pmol/mg F-Asn found in mouse chow is sufficient to intoxicate a Salmonella fraB mutant in mouse models of salmonellosis, and several human foods were found to have F-Asn at this level or higher (fresh apricots, lettuce, asparagus, and canned peaches). Much higher concentrations (11 000-35 000 pmol/mg dry weight) were found in heat-dried apricots, apples, and asparagus. This report reveals possible origins of F-Asn as a nutrient source for Salmonella and identifies foods that could be used together with a FraB inhibitor as a therapeutic agent for Salmonella.

Salmonella FraE, an Asparaginase Homolog, Contributes to Fructose-Asparagine but Not Asparagine Utilization.

Salmonella enterica can utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. This capability has been attributed to five genes in the fra locus. Previously, we determined that mutations in fraB (deglycase), fraD (kinase), or fraA (transporter) eliminated the ability of Salmonella to grow on F-Asn, while a mutation in fraE allowed partial growth. We hypothesized that FraE, a putative periplasmic fructose-asparaginase, converts F-Asn to NH4 + and fructose-aspartate (F-Asp). FraA could then transport F-Asp into the cytoplasm for subsequent catabolism. Here, we report that growth of the fraE mutant on F-Asn is caused by a partially redundant activity provided by AnsB, a periplasmic asparaginase. Indeed, a fraE ansB double mutant is unable to grow on F-Asn. Moreover, biochemical assays using periplasmic extracts of mutants that express only FraE or AnsB confirmed that each of these enzymes converts F-Asn to F-Asp and NH4 + However, FraE does not contribute to growth on asparagine. We tested and confirmed the hypothesis that a fraE ansB mutant can grow on F-Asp, while mutants lacking fraA, fraD, or fraB cannot. This finding provides strong evidence that FraA transports F-Asp but not F-Asn from the periplasm to the cytoplasm. Previously, we determined that F-Asn is toxic to a fraB mutant due to the accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp). Here, we found that, as expected, a fraB mutant is also inhibited by F-Asp. Collectively, these findings contribute to a better understanding of F-Asn utilization by Salmonella IMPORTANCE Salmonella is able to utilize fructose-asparagine (F-Asn) as a nutrient. We recently reported that the disruption of a deglycase enzyme in the F-Asn utilization pathway inhibits the growth of Salmonella in mice and recognized this pathway as a novel and specific drug target. Here, we characterize the first step in the pathway wherein FraE hydrolyzes F-Asn to release NH4 + and F-Asp in the periplasm of the cell. A fraE mutant continues to grow slowly on F-Asn due to asparaginase activity encoded by ansB.

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine.

Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase, and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation owing to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the d-isomer of aspartate) than to the sugar.

A metabolic intermediate of the fructose-asparagine utilization pathway inhibits growth of a Salmonella fraB mutant.

Insertions in the Salmonella enterica fra locus, which encodes the fructose-asparagine (F-Asn) utilization pathway, are highly attenuated in mouse models of inflammation (>1000-fold competitive index). Here, we report that F-Asn is bacteriostatic to a fraB mutant (IC50 19 µM), but not to the wild-type or a fra island deletion mutant. We hypothesized that the presence of FraD kinase and absence of FraB deglycase causes build-up of a toxic metabolite: 6-phosphofructose-aspartate (6-P-F-Asp). We used biochemical assays to assess FraB and FraD activities, and mass spectrometry to confirm that the fraB mutant accumulates 6-P-F-Asp. These results, together with our finding that mutants lacking fraD or the fra island are not attenuated in mice, suggest that the extreme attenuation of a fraB mutant stems from 6-P-F-Asp toxicity. Salmonella FraB is therefore an excellent drug target, a prospect strengthened by the absence of the fra locus in most of the gut microbiota.

Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds.

We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid.

Pyrrolysine-inspired protein cyclization.

The pyrrolysine translational machinery has been extensively explored for the production of recombinant proteins containing a variety of "site-specific" non-canonical amino acids for both in vitro and in vivo biochemical studies. In this study, we report the first use of this technology for the production of branched cyclic proteins with a tadpole-like topology. As a proof of concept, we fused the well-studied RGD peptide to the C terminus of an mCherry reporter protein. Previous studies have shown that cyclization of the RGD peptide enhances its internalization into cells compared to its linear counterpart. The cellular uptake efficiencies of mCherry-cyclo(RGD), mCherry-linear(RGD), and wild-type mCherry determined by flow cytometry follow the trends expected, thereby confirming the feasibility and potential utility of this cyclization approach.

Fructose-asparagine is a primary nutrient during growth of Salmonella in the inflamed intestine.

Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10-/- mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1- SPI2- or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.

Iron(II)-dependent dioxygenase and N-formylamide deformylase catalyze the reactions from 5-hydroxy-2-pyridone to maleamate.

5-Hydroxy-2-pyridone (2,5-DHP) is a central metabolic intermediate in catabolism of many pyridine derivatives, and has been suggested as a potential carcinogen. 2,5-DHP is frequently transformed to N-formylmaleamic acid (NFM) by a 2,5-DHP dioxygenase. Three hypotheses were formerly discussed for conversion of 2,5-DHP to maleamate. Based on enzymatic reactions of dioxygenase (Hpo) and N-formylamide deformylase (Nfo), we demonstrated that the dioxygenase does not catalyze the hydrolysis of NFM but rather that this activity is brought about by a separate deformylase. We report that the deformylase acts both on NFM and its trans-isomer, N-formylfumaramic acid (NFF), but the catalytic efficiency of Nfo for NFM is about 1,400 times greater than that for NFF. In addition, we uncover catalytic and structural characteristics of the new family that the Hpo belongs to, and support a potential 2-His-1-carboxylate motif (HX52HXD) by three-dimensional modeling and site-directed mutagenesis. This study provides a better understanding of 2,5-DHP catabolism.

Channel-forming solvate crystals and isostructural solvent-free powder of 5-hydroxy-6-methyl-2-pyridone.

Crystals of 5-hydroxy-6-methyl-2-pyridone, (I), grown from a variety of solvents, are invariably trigonal (space group R3); these are 5-hydroxy-6-methyl-2-pyridone acetone 0.1667-solvate, C(6)H(7)NO(2)·0.1667C(3)H(6)O, (Ia), and 6-methyl-5-hydroxy-2-pyridone propan-2-ol 0.1667-solvate, C(6)H(7)NO(2)·0.1667C(3)H(8)O, (Ib), and the forms from methanol, (Ic), water, (Id), benzonitrile, (Ie), and benzyl alcohol, (If). They incorporate channels running the length of the c axis that contain extensively disordered solvent molecules. A solvent-free sublimed powder of 5-hydroxy-6-methyl-2-pyridone microcrystals is essentially isostructural. Inversion-related host molecules interact via pairs of N-H...O hydrogen bonds to form R(2)(2)(8) dimers. Six of these dimers form large R(12)(6)(42) puckered rings, in which the O atom of each N-H...O hydrogen bond is also the acceptor in an O-H...O hydrogen bond that involves the 5-hydroxy group. The large R(12)(6)(42) rings straddle the -3 axes and form stacked columns via π-π interactions between inversion-related molecules of (I) [mean interplanar spacing = 3.254 Šand ring centroid-centroid distance = 3.688 (2) Å]. The channels are lined by methyl groups, which all point inwards to the centre of the channels.

Channel-forming solvates of 6-chloro-2,5-dihydroxypyridine and its solvent-free tautomer 6-chloro-5-hydroxy-2-pyridone.

On crystallization from CHCl3, CCl4, CH2ClCH2Cl and CHCl2CHCl2, 6-chloro-5-hydroxy-2-pyridone, C5H4ClNO2, (I), undergoes a tautomeric rearrangement to 6-chloro-2,5-dihydroxypyridine, (II). The resulting crystals, viz. 6-chloro-2,5-dihydroxypyridine chloroform 0.125-solvate, C5H4ClNO(2).0.125CHCl3, (IIa), 6-chloro-2,5-dihydroxypyridine carbon tetrachloride 0.125-solvate, C5H4ClNO(2)..0.125CCl4, (IIb), 6-chloro-2,5-dihydroxypyridine 1,2-dichloroethane solvate, C5H4ClNO2.C2H4Cl2, (IIc), and 6-chloro-2,5-dihydroxypyridine 1,1,2,2-tetrachloroethane solvate, C5H4ClNO2.C2H2Cl4, (IId), have I4(1)/a symmetry, and incorporate extensively disordered solvent in channels that run the length of the c axis. Upon gentle heating to 378 K in vacuo, these crystals sublime to form solvent-free crystals with P2(1)/n symmetry that are exclusively the pyridone tautomer, (I). In these sublimed pyridone crystals, inversion-related molecules form R(2)(2)(8) dimers via pairs of N-H...O hydrogen bonds. The dimers are linked by O-H...O hydrogen bonds into R(4)(6)(28) motifs, which join to form pleated sheets that stack along the a axis. In the channel-containing pyridine solvate crystals, viz. (IIa)-(IId), two independent host molecules form an R(2)(2)(8) dimer via a pair of O-H...N hydrogen bonds. One molecule is further linked by O-H...O hydrogen bonds to two 4(1) screw-related equivalents to form a helical motif parallel to the c axis. The other independent molecule is O-H...O hydrogen bonded to two 4 related equivalents to form tetrameric R(4)(4)(28) rings. The dimers are pi-pi stacked with inversion-related dimers, which in turn stack the R(4)(4)(28) rings along c to form continuous solvent-accessible channels. CHCl3, CCl4, CH2ClCH2Cl and CHCl2CHCl2 solvent molecules are able to occupy these channels but are disordered by virtue of the 4 site symmetry within the channels.